Human glucose-dependent insulin-secreting cell line

• US 6,642,003 B2
• Filed: 08/02/2001
• Issued: 11/04/2003
• Est. Priority Date: 08/02/2001
• Status: Expired due to Fees

• Current Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Original Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Inventors: Perfetti, Riccardo
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/920,868
• Publication Number:
  US 20030113300A1
• Time in Patent Office: 824 Days
• Field of Search: 435/6, 435/458, 435/459, 435/461, 435/366
• US Class Current: 435/6.13
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

Disclosed herein is a novel cell line of human pancreatic cells that secrete insulin in a glucose-dependent manner. The cell line comprises pancreatic cells, such as PANC-1 cells, which are transfected so as to express IDX-1 and cultured in GLP-1. The cell line may be used to investigate the function and development of pancreatic cells, as well as to test the efficacy of drugs that stimulate insulin secretion.

Claims

1. A cell that exhibits a dose-dependent response of insulin secretion when exposed to glucose, the cell comprising an exocrine pancreatic cell transfected with IDX-1 cDNA and cultured in glucagon-like peptide-1 (GLP-1).

2. The insulin-secreting cell of claim 1, wherein the pancreatic cell is a PANC-1 cell.

3. The insulin-secreting cell of claim 1, wherein the pancreatic cell is selected from the group consisting of A, B, D, and F islet cells, epithelial cells of intralobular ducts, acinar cells, centroacinar cells, and basket cells.

4. The insulin-secreting cell of claim 1, wherein the pancreatic cell is selected from the group consisting of AsPc-1, AR42J, BxPc-3, Capan-1, Capan-2, Colo357, HPAC, HPAF, HPAF-II, Hs766T, human insulinoma cell line, MiaPaCa, MIAPaCa-2, PANC-1, Panc89, QGP-1, S2CP9, and T3M4 cells.

5. The insulin-secreting cell of claim 1, wherein the cell is cultured in 1-20 nM GLP-1.

6. The insulin-secreting cell of claim 5, wherein the cell is cultured in 10 nM GLP-1.

7. The insulin-secreting cell of claim 5, wherein the cell is cultured for 24-72 hours in GLP-1.

8. The insulin-secreting cell of claim 1, wherein the cell is cultured in DMEM medium.

9. The insulin-secreting cell of claim 1, wherein the cell is cultured in at least one supplement selected from the group consisting of fetal calf serum, penicillin, and streptomycin.

10. A method of producing insulin-secreting cells that exhibit a dose-dependent response of insulin secretion when exposed to glucose, comprising:

transfecting a line of exocrine pancreatic cells with genetic material selected from the group consisting of IDX-1 DNA, IDX-1 cDNA, and IDX-1 mRNA; and

culturing the cells in glucagon-like peptide-1 (GLP-1).

11. The method according to claim 10, wherein the pancreatic cells comprise a PANC-1 cell line.

12. The method according to claim 10, wherein the pancreatic cells comprise a cell line selected from the group consisting of AsPc-1, AR42J, BxPc-3, Capan-1, Capan-2, Colo357, HPAC, HPAF, HPAF-II, Hs766T, human insulinoma cell line, MiaPaCa, MIAPaCa-2, PANC-1, Panc89, QGP-1, S2CP9, and T3M4 cells.

13. The method according to claim 10, wherein transfecting the cells with genetic material comprises transfecting the cells with IDX-1 cDNA.

14. The method according to claim 13, wherein transfecting the cells with genetic material further comprises transfecting the cells with a pcDNA3 construct comprising wild-type IDX-1 cDNA.

15. The method according to claim 13, wherein transfecting the cells with genetic material further comprises transfecting the cells by a method selected from the group consisting of coprecipitation of DNA with calcium phosphate, lipofection with cationic liposomes, transfection with polyethylenimine, transfection by electroporation, and receptor-mediated endocytosis.

16. The method according to claim 13, the method further comprising selecting cells that are successfully transfected with the genetic material from cells that are not successfully transfected.

17. The method according to claim 16, the method further comprising transfecting the cells with genetic material that encodes aminoglycoside phosphotransferase and culturing the cells in neomycin geneticin sulfate.

18. The method according to claim 16, the method further comprising transfecting the cells with genetic material that encodes hygromycin phosphotransferase and culturing the cells in hygromycin B.

19. The method according to claim 16, the method further comprising transfecting the cells with genetic material that encodes puromycin N-acetyl transferase and culturing the cells in puromycin.

20. The method according to claim 16, the method further comprising transfecting the cells with genetic material that encodes histidinol dehydrogenase and culturing the cells in histidinol.

21. The method according to claim 16, the method further comprising transfecting the cells with genetic material that encodes dihydrofolate reductase and culturing the cells in methotrexate.

22. The method according to claim 10, wherein culturing the cells in GLP-1 comprises culturing the cells in GLP-1 at a concentration of 1-20 nM GLP-1.

23. The method according to claim 10, wherein culturing the cells in GLP-1 comprises culturing the cells in GLP-1 at a concentration of 10 nM GLP-1.

24. The method according to claim 10, wherein culturing the cells in GLP-1 further comprises culturing the cells in GLP-1 for 24-72 hours.

25. The method according to claim 10, wherein culturing the cells in GLP-1 further comprises culturing the cells in GLP-1 at 37°

C. under 95% air and 5% CO₂.

26. The method according to claim 10, wherein culturing cell in GLP-1 further comprises culturing the cells in DMEM medium.

27. The method according to claim 10, wherein culturing cell in GLP-1 further comprises culturing the cells in at least one supplement selected from the group consisting of fetal calf serum, penicillin, and streptomycin.

28. A method of determining the ability of a drug to stimulate cells that exhibit a dose-dependent response of insulin secretion when exposed to glucose to secrete insulin, comprising:

exposing exocrine pancreatic cells that have been transfected with IDX-1 cDNA and cultured in GLP-1 to the drug; and

measuring the amount of insulin secreted by the cells.

29. The method of claim 28, wherein the step of exposing the cells to the drug comprises exposing the cells to a sulfonylurea.

30. The method of claim 28, wherein the step of exposing the cells to the drug comprises exposing the cells to a drug selected from the group consisting of metformin, acarbose, troglitazone, and repaglinide.